PROJECT SUMMARY ? Mapping Technology Development The organization of chromosomes inside the cell nucleus is involved in regulating many genomic processes including gene expression. The folding of chromosomes can be determined using a suite of molecular methods based on the chromosome conformation capture technology. Recent improvements of this basic technology, e.g. Hi-C, now allow analysis of chromosome structure at increasing scale (genome-wide) and resolution (Kb). However, further method optimization and quantitative validation is required before these methods can be used to generate interaction maps of the genome that can serve as a standardized reference maps for the community and for deeper analysis by computational approaches and physical modeling. We will further develop a set of Hi-C-based technologies that when combined will provide detailed information on the conformation of the human genome across all length scales, from nucleosome-level chromatin fibers to nucleus-wide organization of chromosomes. First, we have already developed and fully implemented the Hi-C methodology for genome-wide mapping of the organization of the genome. We will further develop this approach to increase resolution, and to reduce noise, bias and background. Second, we will further develop Micro-C, a new 3C-based method we developed that employs micrococcal nuclease to fragment chromatin. Micro-C provides structural information at the scale of single nucleosomes, and the folding of nucleosomal arrays into higher order assemblies such as 30 nm fibers. This level of chromatin folding is not visible with any other existing 3C-based technology. Pilot interaction maps generated with the different Hi-C-based approaches will be validated and bench marked using a high-throughput imaging platform (HIPMap) developed and operational in the Misteli lab. We will use HIPMap to 1) Validate and refine large scale datasets by determining the contact frequency of mapped interactors in 3D space at the single cell level, 2) Determine cell-to-cell variability of mapped interactions by single cell analysis of the statistical distribution of localization data of mapped interactors. Several hundred probe pairs covering defined domains/interactions (TADs, enhancer/promoter, active/inactive chromatin) on the scale of Kb to Mb will be used at high probe resolution for several thousand cells per probe pair, representing one of the most comprehensive FISH analyses of the 4D nucleome to date. This data will also be used for modeling chromosome conformation described in other components of this application. Once validated, we will use the different Hi-C methods to generate Reference Interaction Maps at the scale of nucleosomes, chromatin fibers, chromosomes and the whole nucleus, in cell populations and in single cells. Maps will be generated for four key biological states representing different conformations during the cell cycle (interphase and mitosis), and during cell differentiation (pluripotent and differentiated states).